EP3654934B1

EP3654934B1 - Oral care compositions and uses thereof

Info

Publication number: EP3654934B1
Application number: EP18765252.4A
Authority: EP
Inventors: Carl MYERS; Guillaume PICQUET; Kathryn Uhrich; Jennifer CHAN WOO; Jeannette Elizabeth MARINE
Original assignee: Rutgers State University of New Jersey; Colgate Palmolive Co
Current assignee: Rutgers State University of New Jersey; Colgate Palmolive Co
Priority date: 2017-08-23
Filing date: 2018-08-22
Publication date: 2023-10-04
Anticipated expiration: 2038-08-22
Also published as: CN111032011A; MX2020001954A; EP3654934A1; MX2021014806A; US20190060210A1; CN111032011B; CA3073444A1; US10744079B2; WO2019040545A1

Description

BACKGROUND

Bacteria attached to an oral surface can cause local and systemic pathology. For example, periodontitis and caries are caused by oral surface bacterial biofilm formation, and are among the most common oral diseases worldwide. Seeding of oral cavity bacteria into the bloodstream can occur during dental procedures and result in serious infections such as endocarditis and prosthetic joint infection.
Document WO2015/157241 relates to oral care compositions comprising substantive polyesters formed from xylitol, polycarboxylic acids (or esters, acid halides or anhydrides thereof) and optionally arginine. The formed polyesters or polyesteramides are active in biofilm inhibition and dissolution to maintain clean teeth.
A need exists for oral care products having an improved anti-bacterial effect to prevent and/or decrease the incidence of local and systemic pathology arising from bacterial attachment to an oral surface. The compositions and methods of the present invention address this need.

BRIEF SUMMARY

In some embodiments, the present invention provides a composition comprising an amphiphilic macromolecule (AM) as defined in the appended claims. The AMs of the present invention are effective in (1) preventing or decreasing oral surface bacterial attachment, (2) stabilizing flavor in an oral care composition, or (3) both preventing or decreasing oral surface bacterial attachment and stabilizing flavor in an oral care composition.

DETAILED DESCRIPTION

The present invention is directed to an oral care composition comprising specific amphiphilic macromolecules selected from compounds A, B, C, D, E and F, and an orally acceptable carrier, for use in a method for inhibiting or preventing bacterial adhesion to an oral cavity surface comprising contacting an oral cavity surface of a mammal with the oral care composition. Compounds A, B, C, D, E and F are as defined in the appended set of claims.
As used herein, the terms "oral composition(s)" or "oral care composition(s)" refer to a composition that is delivered to one or more surfaces of the oral cavity. Generally, during the normal course of use, the composition is not swallowed; rather, it is retained in the oral cavity for a time sufficient to contact one or more surfaces of the oral cavity and provide the desired benefit. Examples of such compositions include, but are not limited to, toothpaste, mouthwash or mouth rinse, oral gel, denture cleanser, and the like.
As used herein, the term "mouthrinse" or "mouthwash" refers to oral care compositions that are substantially liquid in character. In such a preparation the orally acceptable carrier typically has an aqueous phase comprising water or a water and alcohol mixture. Further, in various embodiments, the oral carrier includes a humectant and surfactant as described below. In various embodiments, the alcohol is typically ethyl alcohol.
In some embodiments, the oral care compositions may further include one or more fluoride ion sources, e.g., soluble fluoride salts. A wide variety of fluoride ion-yielding materials can be employed as sources of soluble fluoride in the present compositions. Examples of suitable fluoride ion-yielding materials are found in U.S. Pat. No. 3,535,421, to Briner et al. ; U.S. Pat. No. 4,885,155, to Parran, Jr. et al. and U.S. Pat. No. 3,678,154, to Widder et al. . Representative fluoride ion sources used with the present invention include, but are not limited to, stannous fluoride, sodium fluoride, potassium fluoride, sodium monofluorophosphate, sodium fluorosilicate, ammonium fluorosilicate, amine fluoride, ammonium fluoride, and combinations thereof. In certain embodiments the fluoride ion source includes stannous fluoride, sodium fluoride, sodium monofluorophosphate as well as mixtures thereof. Where the formulation comprises calcium salts, the fluoride salts are preferably salts wherein the fluoride is covalently bound to another atom, e.g., as in sodium monofluorophosphate, rather than merely ionically bound, e.g., as in sodium fluoride.
In other embodiments, the compositions of the present invention may contain anionic surfactants, for example, water-soluble salts of higher fatty acid monoglyceride monosulfates, such as the sodium salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids such as sodium N- methyl N-cocoyl taurate, sodium coco-glyceride sulfate; higher alkyl sulfates, such as sodium lauryl sulfate; higher alkyl-ether sulfates, e.g., of formula CH₃(CH₂)_mCH₂(OCH₂CH₂)_nOS0₃X, wherein m is 6-16, e.g., 10, n is 1-6, e.g., 2, 3 or 4, and X is Na or , for example sodium laureth-2 sulfate (CH₃(CH2)₁₀CH₂(OCH₂CH₂)₂OS0₃Na); higher alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate (sodium lauryl benzene sulfonate); higher alkyl sulfoacetates, such as sodium lauryl sulfoacetate (dodecyl sodium sulfoacetate), higher fatty acid esters of 1,2 dihydroxy propane sulfonate, sulfocolaurate (N-2-ethyl laurate potassium sulfoacetamide) and sodium lauryl sarcosinate. By "higher alkyl" is meant, e.g., C₆-₃o alkyl. In particular embodiments, the anionic surfactant (where present) is selected from sodium lauryl sulfate and sodium ether lauryl sulfate. When present, the anionic surfactant is present in an amount which is effective, e.g., > 0.001% by weight of the formulation, but not at a concentration which would be irritating to the oral tissue, e.g., 1 %, and optimal concentrations depend on the particular formulation and the particular surfactant. In one embodiment, the anionic surfactant is present at from 0.03% to 5% by weight, e.g., 1.5%.
Cationic surfactants may also be included in compositions of the present invention, and can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having one long alkyl chain containing 8 to 18 carbon atoms such as lauryl trimethylammonium chloride, cetyl pyridinium chloride, cetyl trimethylammonium bromide, di-isobutylphenoxyethyldimethylbenzylammonium chloride, coconut alkyltrimethylammonium nitrite, cetyl pyridinium fluoride, and mixtures thereof. Illustrative cationic surfactants are the quaternary ammonium fluorides described in U.S. Pat. No. 3,535,421, to Briner et al. . Certain cationic surfactants can also act as germicides in the compositions.
Illustrative nonionic surfactants that can be used in some embodiments of the present invention can be broadly defined as compounds produced by the condensation of alkylene oxide groups (hydrophilic in nature) with an organic hydrophobic compound which may be aliphatic or alkylaromatic in nature. Examples of suitable nonionic surfactants include, but are not limited to, the Pluronics, polyethylene oxide condensates of alkyl phenols, products derived from the condensation of ethylene oxide with the reaction product of propylene oxide and ethylene diamine, ethylene oxide condensates of aliphatic alcohols, long chain tertiary amine oxides, long chain tertiary phosphine oxides, long chain dialkyl sulfoxides and mixtures of such materials. In a particular embodiment, the composition of the invention comprises a nonionic surfactant selected from polaxamers (e.g., polaxamer 407), polysorbates (e.g., polysorbate 20), polyoxyl hydrogenated castor oils (e.g., polyoxyl 40 hydrogenated castor oil), and mixtures thereof.
Illustrative amphoteric surfactants that can be used in the compositions of the invention include betaines (such as cocamidopropyl betaine), derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be a straight or branched chain and wherein one of the aliphatic substituents contains about 8-18 carbon atoms and one contains an anionic water-solubilizing group (such as carboxylate, sulfonate, sulfate, phosphate or phosphonate), and mixtures of such materials.
Illustrative zwitterionic surfactants that can be used in some embodiments of the present invention include derivatives of aliphatic quaternary ammonium, phosphonium and sulfonium compounds in which the aliphatic radical can be a straight or branched chain and wherein one of the aliphatic substituents contains about 8-18 carbon atoms and one contains an anionic water-solubilizing group (such as carboxy, sulfonate, sulfate, phosphate or phosphonate). The surfactant or mixtures of compatible surfactants can be present in the compositions of the present invention in 0.1% to 5%, in another embodiment 0.3% to 3% and in another embodiment 0.5% to 2% by weight of the total composition.
The oral care compositions of the invention may also include a flavoring agent. Flavoring agents which are used in the practice of the present invention include, but are not limited to, essential oils and various flavoring aldehydes, esters, alcohols, and similar materials, as well as sweeteners such as sodium saccharin. Examples of the essential oils include oils of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, lime, grapefruit, and orange. Also useful are such chemicals as menthol, carvone, and anethole. Certain embodiments employ the oils of peppermint and spearmint. In some embodiments, the flavoring agent is incorporated in the oral composition at a concentration of 0.01 to 1% by weight.
Some AMs help to solubilize the flavoring agent in the oral care composition.
The oral care compositions of the invention also may include one or more chelating agents able to complex calcium found in the cell walls of the bacteria. Binding of this calcium weakens the bacterial cell wall and augments bacterial lysis.
Another group of agents suitable for use as chelating or anti-calculus agents in the present invention are the soluble pyrophosphates. The pyrophosphate salts used in the present compositions can be any of the alkali metal pyrophosphate salts. In certain embodiments, salts include tetra alkali metal pyrophosphate, dialkali metal diacid pyrophosphate, trialkali metal monoacid pyrophosphate and mixtures thereof, wherein the alkali metals are sodium or potassium. The salts are useful in both their hydrated and unhydrated forms. An effective amount of pyrophosphate salt useful in the present composition is generally enough to provide least 0.1 wt. % pyrophosphate ions, e.g., 0.1 to 3 wt 5, e.g., 0.1 to 2 wt %, e.g., 0.1 to 1 wt%, e.g., 0.2 to 0.5 wt%. The pyrophosphates also contribute to preservation of the compositions by lowering the effect of water activity.
The oral care compositions of the invention also optionally include one or more polymers, such as polyethylene glycols, polyvinyl methyl ether maleic acid copolymers, polysaccharides (e.g., cellulose derivatives, for example carboxymethyl cellulose, or polysaccharide gums, for example xanthan gum or carrageenan gum). Acidic polymers, for example polyacrylate gels, may be provided in the form of their free acids or partially or fully neutralized water soluble alkali metal (e.g., potassium and sodium) or ammonium salts. Certain embodiments include 1 :4 to 4: 1 copolymers of maleic anhydride or acid with another polymerizable ethylenically unsaturated monomer, for example, methyl vinyl ether (methoxyethylene) having a molecular weight (M.W.) of about 30,000 to about 1,000,000. These copolymers are available for example as Gantrez AN 139(M.W. 500,000), AN 1 19 (M.W. 250,000) and S-97 Pharmaceutical Grade (M.W. 70,000), of GAF Chemicals Corporation.
Other operative polymers include those such as the 1:1 copolymers of maleic anhydride with ethyl acrylate, hydroxyethyl methacrylate, N-vinyl-2-pyrollidone, or ethylene, the latter being available for example as Monsanto EMA No. 1 103, M.W. 10,000 and EMA Grade 61, and 1 : 1 copolymers of acrylic acid with methyl or hydroxyethyl methacrylate, methyl or ethyl acrylate, isobutyl vinyl ether or N-vinyl-2-pyrrolidone.
Suitable generally, are polymerized olefinically or ethylenically unsaturated carboxylic acids containing an activated carbon-to-carbon olefinic double bond and at least one carboxyl group, that is, an acid containing an olefinic double bond which readily functions in polymerization because of its presence in the monomer molecule either in the alpha-beta position with respect to a carboxyl group or as part of a terminal methylene grouping. Illustrative of such acids are acrylic, methacrylic, ethacrylic, alpha-chloroacrylic, crotonic, beta-acryloxy propionic, sorbic, alpha-chlorosorbic, cinnamic, beta-styrylacrylic, muconic, itaconic, citraconic, mesaconic, glutaconic, aconitic, alpha-phenylacrylic, 2-benzyl acrylic, 2-cyclohexylacrylic, angelic, umbellic, fumaric, maleic acids and anhydrides. Other different olefinic monomers copolymerizable with such carboxylic monomers include vinylacetate, vinyl chloride, dimethyl maleate and the like. Copolymers contain sufficient carboxylic salt groups for water-solubility.
A further class of polymeric agents includes a composition containing homopolymers of substituted acrylamides and/or homopolymers of unsaturated sulfonic acids and salts thereof, in particular where polymers are based on unsaturated sulfonic acids selected from acrylamidoalykane sulfonic acids such as 2-acrylamide 2 methylpropane sulfonic acid having a molecular weight of about 1,000 to about 2,000,000, described in U.S. Pat. No. 4,842,847, Jun. 27, 1989 Zahid.
Another useful class of polymeric agents includes polyamino acids, particularly those containing proportions of anionic surface-active amino acids such as aspartic acid, glutamic acid and phosphoserine, as disclosed in U.S. Pat. No. 4,866,161 Sikes et al. .
In preparing oral care compositions, it is sometimes necessary to add some thickening material to provide a desirable consistency or to stabilize or enhance the performance of the formulation. In certain embodiments, the thickening agents are carboxyvinyl polymers, carrageenan, xanthan gum, hydroxyethyl cellulose and water soluble salts of cellulose ethers such as sodium carboxymethyl cellulose and sodium carboxymethyl hydroxyethyl cellulose. Natural gums such as karaya, gum arabic, and gum tragacanth can also be incorporated. Colloidal magnesium aluminum silicate or finely divided silica can be used as component of the thickening composition to further improve the composition's texture. In certain embodiments, thickening agents in an amount of about 0.5% to about 5.0% by weight of the total composition are used.
The pH of the compositions of the present invention is generally between about 4.5 and about 10. The pH can be controlled with acid (e.g., citric acid or benzoic acid) or base (e.g., sodium hydroxide) or buffered (with sodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogen phosphate, or sodium dihydrogen phosphate, for example).
Colorants among those useful herein include pigments, dyes, lakes and agents imparting a particular luster or reflectivity such as pearling agents. In various embodiments, colorants are operable to provide a white or light-colored coating on a dental surface, to act as an indicator of locations on a dental surface that have been effectively contacted by the composition, and/or to modify appearance, in particular color and/or opacity, of the composition to enhance attractiveness to the consumer. Any orally acceptable colorant can be used, including FD&C dyes and pigments, talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, magnesium aluminum silicate, silica, titanium dioxide, zinc oxide, red, yellow, brown and black iron oxides, ferric ammonium ferrocyanide, manganese violet, ultramarine, titaniated mica, bismuth oxychloride, and mixtures thereof. One or more colorants are optionally present in a total amount of 0.001 wt.% to 10 wt.%, for example 0.01 wt.% to 5 wt.% or 0.1 wt.% to 1 wt.%.
In some embodiments, the compositions of the present invention comprise a tartar control (anticalculus) agent. Tartar control agents among those useful herein include salts of any of these agents, for example their alkali metal and ammonium salts: phosphates and polyphosphates (for example pyrophosphates), polyaminopropanesulfonic acid (AMPS), polyolefin sulfonates, polyolefin phosphates, diphosphonates such as azacycloalkane-2,2-diphosphonates (e.g., azacycloheptane-2,2-diphosphonic acid), N-methyl azacyclopentane-2,3-diphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) and ethane-1-amino-1,1-diphosphonate, phosphonoalkane carboxylic acids and. Useful inorganic phosphate and polyphosphate salts include monobasic, dibasic and tribasic sodium phosphates, sodium tripolyphosphate, tetrapolyphosphate, mono-, di-, tri- and tetrasodium pyrophosphates, sodium trimetaphosphate, sodium hexametaphosphate and mixtures thereof.
In further embodiments, the oral compositions of the present invention comprise a saliva stimulating agent useful, for example, in amelioration of dry mouth. Any orally acceptable saliva stimulating agent can be used, including without limitation food acids such as citric, lactic, malic, succinic, ascorbic, adipic, fumaric and tartaric acids, and mixtures thereof. One or more saliva stimulating agents are optionally present in saliva stimulating effective total amount.
In yet other embodiments, the oral compositions of the present invention comprise a nutrient. Suitable nutrients include vitamins, minerals, amino acids, and mixtures thereof. Vitamins include Vitamins C and D, thiamine, riboflavin, calcium pantothenate, niacin, folic acid, nicotinamide, pyridoxine, cyanocobalamin, para-aminobenzoic acid, bioflavonoids, and mixtures thereof. Nutritional supplements include amino acids (such as L-tryptophane, L-lysine, methionine, threonine, levocamitine and L-carnitine), lipotropics (such as choline, inositol, betaine, and linoleic acid), and mixtures thereof.
The compositions as defined in the claims are useful in a method of treating conditions associated with the presence of oral bacteria comprising providing an oral composition in accordance to the claims, and applying the oral composition to the oral cavity of the mammalian subject. The method comprises repeating the application of the composition multiple times until the desired anti-bacterial and/or antiinflammatory effects are achieved in the subject.
The invention provides composition as described in the appended claims use in a method for inhibiting or preventing bacterial adhesion to an oral cavity surface of a mammal. In some embodiments, the compounds described herein (e.g. Compounds A to D) can be prepared in accordance with the methods described in US 2011/0229416 .
Exemplary embodiments of the present disclosure will be illustrated by reference to the following examples.

EXAMPLES

Example 1

Compound A was prepared as follows. CDI (82.0 mg, 0.51 mmol) was added to a solution containing Compound E (1.50 g, 0.25 mmol) in anhydrous DMF (10.5 mL).
The reaction mixture was stirred for 1.5 h at room temperature under a N₂ atmosphere and added to a solution of 2-aminoethyl dihydrogen phosphate (47.5 mg, 0.34 mmol) in water (10.5 mL) containing Et₃N (35 µL), and the mixture was stirred at room temperature for 20 h. The reaction mixture was concentrated in vacuo. The resulting solids were dissolved in DCM and washed once with 0.1 M HCl (aq). The organic layer was washed once with saturated NaCl (aq) solution and dried over anhydrous MgSO_4. The solids were removed by filtration and the solvent was removed from the filtrate in vacuo. The resulting crude oil was dissolved in DCM and precipitated into cold Et₂O. Compound A was isolated via centrifugation at 3500 rpm for 5 min and decanting the supernatant. The pellet was washed with Et₂O (50 mL × 3) and isolated via centrifugation.
A similar method was used to prepare Compound B, using Compound F as the starting material.
Specifically, Compound F was activated with CDI followed by treatment with 2-aminoethylphosphate to afford Compound B. Compounds C and D can be prepared using similar methods, using Compounds G and H, respectively, as the starting materials.
Compounds G and H can be prepared in accordance with the methods described in Tian, L., Novel Amphiphilic Macromolecules for Drug Delivery Applications: Design, Synthesis and Characterization. Rutgers University: 2004.
Compounds E and F can be prepared in accordance with the methods described in Tian et al., Amphiphilic Scorpion-like Macromolecules: Design, Synthesis, and Characterization. Macromolecules 2004, 37 (2), 538-543.

Example 2: Anti-Attachment Assay

Day 1: Over-Night Preparation: Fresh microbial cultures of Actinomyces viscosus (ATCC#43146) & Streptococcus oralis (ATCC#35037) were grown.

Day 2: Treatment:

An HAP coated MBEC Lid (Innovotech Catalog #19132) was placed into its corresponding 96-well plate containing simple solutions or formulations containing the amphiphilic macromolecules for 60 minutes at 37 °C. After this time, the plate was rinsed to remove excess or loosely bound material by dipping the MBEC lid in 1/4 concentrated TSB 10 times. The 1/4 TSB was then changed and the rinsing procedure repeated for a total of three rinsing cycles. Equal volume over-night cultures of A. viscosus & S. oralis were mixed together and the O.D. of the cultures was adjusted to 1.00 at 610 nm. To each of the 96 wells and 180 µl of bacteria solution was added, and the plate was incubated 3-4 hours at 37 °C. Attached bacteria were removed by placing the MBEC lid into fresh 1/4 TSB and sonicating for 2 x 2 minutes, rotating the plate 180° between sonication rounds. A representative sample of 100 µL was removed from each well and transferred to a black, clear-bottom plate (Corning Ref# 3904). Bacteria count is measured by ATP bioluminescence using BacTiter-Glo Microbial Cell Viability Assay (Promega Ref# G8231) as per the manufacturer's instructions.
Table 1 (below) describes the results of the above-described experiment. Table 1

Compound % Reduction

A 65.0

B 46.2

C 42.3

D 40.2

E 47.4

F 25.1
Generally, a correlation between % reduction of bacterial adhesion and how much material was retained after rinsing on HAP-coated surfaces was observed. This data is described in Table 2 (below). Table 2

Compound % Retained* % Retained** % Reduction of Bacterial Adhesion

A 74 100 51.0

B 0 14 26.2

C 0 69 17.6

D 0 0 14.3

E 0 60 75.9

F 0 0 77.9

^∗ HAP-coated QCM-D chips
^∗∗ HAP-coated QCM-D chips (w/ Phosphate buffer)

Example 3: Flavor stabilization assay

Peppermint flavor oil was dissolved in propylene glycol, a typical procedure in the production of commercial mouthwash formulations. Exemplary compositions of the present invention comprising compounds described herein at concentrations of 1 w/w% and 2 w/w %; 7 w/w% propylene glycol; and 0.12 w/w% peppermint flavor are evaluated for turbidity in order to determine which compound(s) exhibited the greatest ability to solubilize flavor. Table 3 (below) describes the results of these evaluations. Table 3

Compound Concentration (w/w %) Transmittance (normalized)

Water N/A 1

Poloxomer 407 1 0.99

F 1 0

2 0

D 1 0.019

2 0

E 1 0.37

2 0.78

A 1 0.19

2 0.90

Example 4

Compound A and Compound E are complexed with flavor and their ability to provide anti-attachment benefits, with and without flavor, is evaluated. Table 4 (below) describes the results of these evaluations, which demonstrate that the presence of a hydrophobic ingredient (e.g. a flavor) does not meaningfully compromise the anti-attachment efficacy of exemplary compounds of the present invention. Table 4

Compound % Reduction

A 51.0

A + Flavor 48.5

E 75.9

E + Flavor 78.8

Example 5

Hydroxyapatite discs (12 mm) are soaked in clarified saliva at 37 °C for 1 hour. After this time, the discs are rinsed and treated with 1.5 mL of a 1 wt% solution containing the materials for 1 hour. The discs are rinsed 3 times and then set in 1.5 mL bacteria solution (50/50 A. viscosus / S. oralis, with an O.D. = 2 @ 610 nm) for 3 hours. Discs are rinsed, and then stained with SYTO 9 dye (3.34 mM, Invitrogen LIVE/DEAD BacLIGHT (TM) Bacterial Viability Kit (L7012), with a concentration of 6 uL/mL. Discs are then rinsed twice with quarter strength TSB buffer for final imaging.

Example 6

Table 5 (below) describes the formula of an exemplary oral care composition of the present invention.

Ingredient	Wt. %
Amphiphilic Macromolecule (e.g. Compounds A-H)	0-2
Antibacterial	0.01 - 0.1
Sodium saccharin	0.005 - 0.2
Sodium benzoate	0.01 - 0.1
Potassium sorbate	0.01 - 1
Glycerin	0 - 15
Sorbitol	0 - 15
Poloxamer 407	0 - 2
Propylene Glycol	0 - 7
Flavor	0.05 - 1
Color	0.0001-0.005
Water	Q.S.

Compositions of the present invention, including the exemplary composition described in Table 5 (above), can be prepared according to methods generally known to those skilled in the art.

Claims

An oral care composition comprising a compound selected from

and
and
an orally acceptable carrier,

for use in a method for inhibiting or preventing bacterial adhesion to an oral cavity surface comprising contacting an oral cavity surface of a mammal with the oral care composition.
The oral care composition for use according to claim 1, wherein the compound is:
The oral care composition for use according to claim 1, wherein the compound is:
The oral care composition for use according to claim 1, wherein the compound is:
The oral care composition for use according to claim 1, wherein the compound is:
The oral care composition for use according to claim 1, wherein the compound is:
The oral care composition for use according to claim 1, wherein the compound is:
The oral care composition for use according to any foregoing claims, wherein the oral care composition is in a form selected from a mouthwash, a toothpaste and an oral spray.